The present invention relates to compositions and methods for detecting mycoplasma infection in the commercial poultry industry. More particularly, the invention relates to the identification and purification of antigens and antisera that are capable of detecting and distinguishing infection caused by Mycoplasma gallisepticum and Mycoplasma synoviae and the use of these antigens in serological assays. Furthermore, a method of producing clones which express the antigenic portions of the antigens is provided.
The p64, p56, p26, p41, and p22 antigens described herein have been deposited with the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC designation Nos. ATCC 40749, ATCC 40748, ATCC 40746, ATCC 40747 and ATCC 40745, respectively. The deposits will be maintained for a period of thirty years, for five years after the last request for the antigen, or for the enforceable life of the U.S. Patent, whichever is longer. Should the deposits become non-viable, they will be replaced by Applicants. Assurance of access to the deposits as determined by the Commissioner under 37 CFR .sctn.1.14 is provided for. All restrictions on the availability of the deposits to the public will be irrevocably removed upon the granting of a patent.
Mycoplasma gallisepticum, which can cause respiratory disease in chickens, turkeys, and other species of birds, is one of the most important pathogens affecting the poultry industry. Chickens and turkeys infected with M. gallisepticum show a wide variety of clinical signs ranging from asymptomatic to chronic respiratory disease. Common symptoms include rales, nasal discharge, coughing, reduced feed intake, weight loss, and decline in egg production. Airsacculitis in chickens and turkeys related directly or indirectly to M. gallisepticum infection is a significant cause of condemnations of carcasses at the processing plant. If breeding stock are infected with M. gallisepticum, the typical action is depopulation to prevent egg transmission to the progeny.
Mycoplasma synoviae infection in aves causes less debilitating symptoms than M. gallisepticum infection. Clinical manifestation of the disease often does not occur. However, lameness with or without generalized disease and respiratory symptoms can reduce optimal production, increase food conversion rates, and cause downgrading of carcasses at the processing plant. Breeder flocks infected with M. synoviae might be eradicated.
Poultry breeders and producers need to maintain their flocks free of pathogenic mycoplasmas, particularly M. gallisepticum. Because the infection of poultry with either M. gallisepticum or M. synoviae is often subclinical, screening for signs of infection by these mycoplasmas is usually done by serology. Optimally, this screening process should detect infection in its early stages. The serological test should be sensitive and specific, and identify which mycoplasma species is causing the infection without giving rise to false positive results.
Presently, flocks are screened for antibodies to M. gallisepticum or M. synoviae by the serum plate agglutination (SPA) test. The SPA test primarily measures IgM and is able to detect antibody in the serum within a week of infection. Unfortunately, the SPA test is prone to false positive results (Boyer et al., Avian Dis., 4:546-547 (1960); Bradbury et al., J. Hyg., 70:267-278 (1971); Avakian et al. Avian Dis., 32:262-272 (1988).
One cause of false positive reactions in the SPA tests, as well as in enzyme-linked immunosorbent assays (ELISA's), stems from the antigenic relationship between M. gallisepticum and M. synoviae; some of the antigens of M. gallisepticum and M. synoviae share common epitopes as shown by immunoblotting. In addition to these cross-reactions, strong and frequent positive SPA and ELISA reactions occur when testing chicken antisera to Frey's medium with 12% swine serum (FMS); this result suggests that reactions to medium components contribute to false positive SPA and ELISA serology. Because birds in the field, especially breeders, often receive numerous oil-emulsion vaccines, they are likely to be simultaneously immunized with medium components that were incorporated into the vaccine along with the pathogenic agent. Thus, antisera from poultry often have antibodies directed towards medium components that are likely to cause false positive SPA and ELISA reactions.
Positive SPA test results usually are confirmed by the hemagglutination-inhibition (HI) test, which primarily measures IgG. The HI test, although specific, lacks sensitivity and seldom detects antibody in the serum until two to three weeks after the initiation of infection (Kleven, J. Vet. Res. , 36:563-564 (1975); Kleven et al., Avian Dis., 32:731-741 (1988)). Also, the typical M. gallisepticum antigen strains used to make HI antigen may not be antigenically similar enough to detect antibodies to some of the atypical M. gallisepticum strains.
Enzyme-linked immunosorbent assays (ELISA's) have been developed for M. gallisepticum and for M. synoviae. However, these ELISA tests suffer from poor sensitivity and/or specificity and are prone to false positive reactions.
Thus, the conventional system used to serodiagnose mycoplasma infections in poultry is less than adequate. These various serological tests are often combined with tracheal culturing to produce a final diagnosis. However, it is often difficult to isolate M. gallisepticum and M. synoviae in cultures from flocks that are concurrently infected with one or more nonpathogenic mycoplasma species such a M. gallinarum, M. gallinaceum, M. pullorum, M. gallopavonis, or Acholeolasma laidlawii. These nonpathogenic species usually overgrow the slower growing pathogenic species in vitro. In rare cases, M. gallisepticum isolates have taken as long as three weeks to show signs of growth in broth medium. Once isolated, mycoplasmas are identified by a fluorescent antibody technique. These fluorescent antibody reagents are not available commercially; therefore, laboratory personnel must prepare these reagents themselves.
Using these procedures, it takes one to three weeks for experienced laboratory diagnosticians to determine which, if any, species of Mycoplasma are infecting a flock. However, a breeder company can experience significant economic loss each day that M. gallisepticum infection in a breeder flock is suspected but unconfirmed. Therefore, there exists a need for reliable, sensitive, and specific serodiagnostic tests that can rapidly detect and differentiate between M. gallisepticum and M. synoviae infection.